Detecting And Modelling Conformational States Of The Proton Channel With Voltage-Clamp Fluorometry

The permeation of protons through the voltage-gated proton channel, Hv1, is regulated by the voltage-dependent movement of a transmembrane (S4) helix containing positively charged arginine residues. To examine the movement of this helix with voltage-clamp fluorometry (VCF) we conjugated an environmentally sensitive fluorophore (TAMRA-MTS) to a cysteine residue (E241C) introduced at the extracellular end of the S4 helix of the Ci-Hv1 proton channel expressed in Xenopus laevis oocytes. In previous work we found that the voltage-dependent fluorescence at this position is largely due to quenching by nearby aromatic amino acids. However, the intensity of this fluorescence reporter exhibited complex kinetics and a non-monotonic voltage dependence, even though the S4 helix is believed to move monotonically in response to membrane depolarization. To reconcile these results, we developed both continuum and 3-state transition models in which unidirectional movement of the S4 helix across the membrane causes the fluorescent reporter to first approach towards, then move away from the quenching aromatic residues. Both types of model can readily describe the time and voltage dependence of the fluorescent reporter, suggesting that the complex, non-monotonic VCF signal is entirely consistent with a simple monotonic movement of the S4 helix provided that intermediate positions between the resting and activated states are relatively stable.